p120 is now well established as a key regulator of classical cadherins and a modulator of Rho- GTPases. At cell-cell junctions, p120 is essential for cadherin stability, and appears to control cell-cell adhesion in part by regulating cadherin stability at the cell surface. Other lines of evidence indicate roles in a variety of Rho-mediated activities that control cell morphology, motility, and proliferation. These observations imply critical roles for p120 in cell-cell adhesion and tumor progression. For all known p120 activities aside from cadherin stabilization, regulation is critically dependant on the N-terminal 300 amino acid segment termed the "regulatory domain". p120 mutants lacking this region (e.g, p120-4 isoforms) are dominant active or dominant negative, depending on the action being assayed (e.g, cell-cell adhesion vs. cell motility). It is increasingly evident that the regulatory domain is required for coupling to Rho, which may in turn account for many of the effects of p120 in cells. The regulatory domain also contains the majority of approximately 16 tyrosine (Y) and serine/threonine (S/T) phosphorylation sites, and interacts with several tyrosine-kinases (e.g, PER, Fyn, Yes) and -phosphatases (eg, PTPa, DEP1, SHP1). Collectively, these observations suggest several potential mechanisms for signaling input and output that are likely to modulate p120 and cadherin activity, and outline what we believe to be the mechanical underpinnings of p120 regulation. Nonetheless, it is not yet clear how the N-terminus controls p120 activity or and/or coupling to Rho. Our working hypothesis is that most of p120's key roles in adhesion and cancer are regulated via protein-protein interactions and post-translational modifications of its regulatory domain. To address this issue in vitro and in vivo, we have generated a powerful p120 siRNA knockdown/substitution model system, mapped the major p120 phosphorylation sites, generated panels of p120 phospho-specific antibodies, and created a p120 conditional knockout mouse. To clarify the role of the regulatory domain, we propose in aim 1 to molecularly dissect the region by uncoupling key p120 binding partners and by structure-function analyses of phosphorylation site mutants. In aim 2, we will use our novel p120 phospho-specific antibodies to identify and study the upstream signaling pathways and direct-acting upstream phospho-effectors of p120 function. Aim 3 is to develop a strategy for determining the molecular mechanism by which p120 regulates RhoA. It examines the phenotypic consequences of fibroblast targeted p120 ablation in vitro and in our conditional KO mouse.